A composition for improving bone density and use thereof

Abstract

The application discloses a composition for improving bone density and application thereof. The composition comprises goat milk casein peptides, collagen tripeptides, sea cucumber peptides and osteopontin, can achieve the effect of improving bone density and preventing and treating osteoporosis, and has broad application prospects in promoting calcium absorption by taking into account functionality and nutrition.

Description

Technical Field

[0001] This invention belongs to the field of compositions, and particularly relates to a composition that helps improve bone density and its application. Background Technology

[0002] Osteoporosis, a silent killer threatening the health of middle-aged and elderly people, is characterized by bone loss, destruction of bone microstructure, and decreased bone density, which are closely related to the imbalance of calcium metabolism in the body. Calcium absorption in the intestine depends on the synergistic action of multiple transport mechanisms, mainly including three key steps: the transcellular pathway, the paracellular pathway, and basolateral calcium efflux. In the transcellular pathway, calcium ions in the intestinal lumen first enter the intestinal epithelial cells through calcium ion channels on the apical membrane (such as TRPV5 and TRPV6), and are then transported to the basolateral side in the cytoplasm by calcium-binding proteins (such as Calbindin-D9k). In the paracellular pathway, calcium bound to peptides can be transported via peptide transporters (such as PepT1). Finally, calcium ions reaching the basolateral side need to be actively pumped out of the cells by the sodium-calcium exchanger (NCX-1) and the plasma membrane calcium pump protein (PMCA1b) to enter the bloodstream. These mechanisms together constitute a sophisticated network for intestinal calcium absorption, maintaining calcium homeostasis in the body.

[0003] Currently, the core strategy for clinical intervention in osteoporosis is calcium supplementation combined with vitamin D to promote intestinal calcium absorption. However, traditional calcium supplements (such as calcium carbonate and calcium phosphate) suffer from poor solubility and low bioavailability, and are prone to causing constipation and gastrointestinal discomfort. Studies have shown that organic calcium and amino acid calcium have higher calcium utilization rates and are more beneficial for improving osteoporosis. Although vitamin D can partially improve calcium absorption, its effect is limited by individual metabolic differences and the potential risk of hypercalcemia. While calcium absorption-promoting drugs (such as bisphosphonates and RANKL inhibitors) introduced in recent years can inhibit bone loss, long-term use may be accompanied by serious side effects such as osteonecrosis of the jaw and atypical femoral fractures, failing to fundamentally improve the efficiency of active calcium absorption.

[0004] Bioactive peptides play a key role in improving calcium absorption efficiency. Small molecule peptides (such as casein phosphopeptides and collagen peptides) can synergistically enhance calcium bioavailability through the following multiple pathways: (1) Forming soluble calcium-peptide complexes: Carboxyl groups, phosphoserine residues, etc. in the peptide chain bind to calcium ions, preventing them from precipitating in the alkaline environment of the intestine and maintaining the dissolved state of calcium ions; (2) Activating paracellular transport pathways: Peptide-calcium complexes can be directly recognized by peptide transport proteins on the apical membrane of intestinal epithelial cells and transported into the cell, circumventing the saturation limitation of traditional calcium ion channels.

[0005] Therefore, there is an urgent need to develop a safe, efficient, and multi-target synergistic calcium absorption-promoting composition to provide new ideas for improving bone density and preventing osteoporosis. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to propose a composition that helps improve bone density and its applications. By rationally combining natural absorption-promoting active peptides, specifically sheep milk casein peptides, collagen tripeptides, sea cucumber peptides, and osteopontin, this invention overcomes the limitations of traditional calcium supplements at the molecular level, achieving a significant effect in promoting calcium absorption. This composition combines functionality and nutrition, showing broad application prospects in improving bone density and preventing osteoporosis.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a composition that helps improve bone density, the composition comprising sheep milk casein peptides, collagen tripeptides, sea cucumber peptides, and osteopontin in a mass ratio of 20-26:8-10:25-50:17-43, or for example 25:8:50:17, 24:10:31:35, 23:8:26:43, 22:9:35:34, 20:8:32:40, 20:10:40:30, or 26:8:25:41.

[0008] Furthermore, the mass ratio of the sheep milk casein peptide, collagen tripeptide, sea cucumber peptide, and osteopontin is 20-23:9-10:35-40:30-35.

[0009] In a second aspect, the present invention provides a product comprising the composition described in the first aspect.

[0010] Furthermore, the product is a health food or a medicine, and the product also includes excipients acceptable to health foods or medicines.

[0011] Furthermore, the product comprises the composition described in the first aspect and a calcium source; Preferably, the calcium source includes organic calcium and amino acid calcium; More preferably, the calcium source includes calcium lactate, calcium glycinate, and / or calcium aspartate; for example, the calcium source includes calcium lactate, calcium glycinate, and calcium aspartate in a mass ratio of 1-3:1-3:1-3, such as 1:1:1, 1:1:2, 1:1:3, 1:2:1, 1:2:2, 1:2:3, 1:3:1, 1:3:2, or 1:3:3.

[0012] Thirdly, the present invention provides the use of the composition described in the first aspect in the preparation of products capable of promoting calcium absorption.

[0013] Furthermore, the application includes at least one of the following: (1) Application in the preparation of products that can increase blood calcium levels; (2) Application in the preparation of products that can increase bone calcium levels; (3) Application in the preparation of products that can reduce fecal calcium levels.

[0014] Furthermore, the application includes at least one of the following: (1) Application in the preparation of products that can increase the levels of intestinal calcium channel proteins TRPV5 and / or TRPV6; (2) Application in the preparation of products that can enhance the calcium-binding protein Calbindin-D9k; (3) Application in the preparation of products that can increase the level of peptide transporter PepT1; (4) Application in the preparation of products that can increase the mRNA expression levels of intestinal sodium-calcium exchanger 1 and plasma membrane calcium pump protein.

[0015] Furthermore, the calcium is provided by calcium lactate, calcium glycinate, and / or calcium aspartate.

[0016] Furthermore, the calcium is composed of calcium lactate, calcium glycinate, and calcium aspartate in a mass ratio of 1-3:1-3:1-3, for example, 1:1:1, 1:1:2, 1:1:3, 1:2:1, 1:2:2, 1:2:3, 1:3:1, 1:3:2, or 1:3:3.

[0017] Fourthly, the present invention provides the use of the composition described in the first aspect in the preparation of products that can promote the improvement of bone density and / or help prevent osteoporosis.

[0018] Compared with the prior art, the present invention has the following advantages: (1) This invention demonstrates that the combined intervention of sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin can help the body absorb and utilize calcium, thereby helping to improve bone density and prevent osteoporosis. Moreover, sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin have a synergistic effect.

[0019] (2) The composition of the present invention can synergistically increase the serum and bone calcium levels of osteoporotic mice, reduce the calcium content in mouse feces, and also promote calcium absorption, improve bone density and prevent osteoporosis by increasing the levels of intestinal calcium ion channel proteins TRPV5, TRPV6, calcium binding protein Calbindin-D9k, peptide transporter PepT1, and increasing the mRNA expression levels of intestinal sodium-calcium exchanger 1 (NCX-1) and plasma membrane calcium pump protein (PMCA1b). Attached Figure Description

[0020] Figure 1 The effects of each composition on serum calcium levels in mice are shown in the examples.

[0021] Figure 2The effects of each composition on bone calcium levels in mice are shown in the examples.

[0022] Figure 3 The effects of each composition on mouse fecal calcium levels are shown in the examples.

[0023] Figure 4 The effects of each composition in the examples on the expression levels of mouse intestinal calcium channel proteins TRPV5 and TRPV6 mRNA are shown.

[0024] Figure 5 The effects of each composition in the examples on the expression level of Calbindin-D9k mRNA in the mouse intestine are shown.

[0025] Figure 6 The effects of each composition in the examples on the mouse intestinal peptide transporter PepT1 are shown.

[0026] Figure 7 The effects of each composition in the examples on the mRNA expression levels of mouse intestinal sodium-calcium exchanger 1 (NCX-1) and plasma membrane calcium pump protein (PMCA1b) are shown. Detailed Implementation

[0027] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0028] In the examples, sheep milk casein peptides were purchased from Xi'an Darwen Biotechnology Co., Ltd., collagen tripeptides and sea cucumber peptides were purchased from Hainan Huayan Collagen Technology Co., Ltd., goose muscle peptides were purchased from Hainan Huatai Biotechnology Co., Ltd., osteopontin was purchased from Arla AG, Denmark, calcium carbonate was purchased from Beijing Jinpukang Food Technology Co., Ltd., calcium lactate was purchased from Beijing Jinpukang Food Technology Co., Ltd., calcium glycine was purchased from Beijing Jiuxing Chemical Products Co., Ltd., and calcium aspartate was purchased from Sanya Baitai Biotechnology Co., Ltd.

[0029] Example 1. Screening of raw materials for the composition formulation The Caco-2 cell model is a human clonal colon adenocarcinoma cell line with a structure and function similar to differentiated small intestinal epithelial cells. It possesses structures such as microvilli and contains enzyme systems associated with the brush border epithelium of the small intestine, making it suitable for simulating in vivo intestinal transport experiments. This invention reflects the calcium absorption-promoting efficiency of the composition by measuring the calcium content in Caco-2 cells after intervention with an active substance (composition) that promotes calcium absorption.

[0030] Caco2 cells were seeded in the upper chambers of 6-well Transwell plates (density: 1 × 10⁻⁶). 5 The culture medium was changed every other day until day 21. The resistance value was measured using a resistance meter, and the transepithelial electrical resistance (TEER) value was calculated according to the formula. When TEER > 400 Ω•cm2, the cells differentiated into a complete monolayer, and the next step of osmotic absorption assay could be performed.

[0031] TEER = (Measured resistance value - Blank resistance value) × Surface area of ​​monolayer film Fully differentiated Caco2 monolayers were washed twice with preheated HBSS (pH 7.2). 1.5 mL of HBSS was added to the upper chamber of a Transwell plate, and 2 mL to the lower chamber, and the plates were equilibrated at 37°C for 30 min. Subsequently, the HBSS in the upper chamber was replaced with different peptides and a 3 mM calcium source for subsequent experiments. The cell monolayer was gently washed twice with HBSS. Then, 3 mM HEPES buffer (calcium source) was added to the top side, and calcium transport buffer was added to the bottom side. After incubation for 4 h, 1 mL of sample was taken from the outside of the bottom side and mixed with 1 mL of calcium transport buffer. Calcium content was measured using a calcium content assay kit, and calcium transport was calculated using the following formula.

[0032] Wherein, Bn represents the total calcium transport in 2.5 mL of equilibrium salt solution on the substrate side of each well at each selected time point (4 h) (unit: μg / well). An represents the calcium ion concentration measured by the calcium content detection kit (unit: μg / mL). n is the time point corresponding to 4 h.

[0033] As shown in Table 1, sheep milk casein peptide, collagen tripeptide, sea cucumber peptide, goose muscle peptide, and osteopontin all have the activity of increasing calcium uptake by Caco2 cells.

[0034] Taking into account factors such as the bioactivity, economic cost, sensory characteristics, and safety of the raw materials, four components—sheep milk casein peptide, collagen tripeptide, sea cucumber peptide, and osteopontin—were selected as raw materials for subsequent composition formulation research.

[0035] Table 1. Effects of formulation ingredients on calcium uptake by CaCO2 cells

[0036] Example 2. Effect of the composition on promoting calcium uptake in Caco2 cells Weigh the raw materials according to the following proportions, and mix them in a double helix conical mixer for 15-30 minutes to obtain the composition. The formulations of each composition are as follows: Composition I Ingredients: 25 g sheep milk casein peptide, 8 g collagen tripeptide, 30 g sea cucumber peptide, 37 g osteopontin; Composition II Ingredients: 24 g sheep milk casein peptide, 10 g collagen tripeptide, 31 g sea cucumber peptide, 35 g osteopontin; Composition III Ingredients: 23 g sheep milk casein peptide, 8 g collagen tripeptide, 26 g sea cucumber peptide, 43 g osteopontin; Composition IV Ingredients: 22 g sheep milk casein peptide, 9 g collagen tripeptide, 35 g sea cucumber peptide, 34 g osteopontin; Composition V ingredients: 20 g sheep milk casein peptide, 8 g collagen tripeptide, 32 g sea cucumber peptide, 40 g osteopontin; Composition VI Ingredients: 20 g sheep milk casein peptide, 10 g collagen tripeptide, 40 g sea cucumber peptide, 30 g osteopontin; Composition VII Ingredients: 26 g sheep milk casein peptide, 8 g collagen tripeptide, 25 g sea cucumber peptide, 41 g osteopontin; Comparative Example 1 Ingredients: 100 g of sheep milk casein peptides; Comparative Example 2 Raw Material: Collagen tripeptide 100 g; Comparative Example 3 Ingredients: 100 g of sea cucumber peptides; Comparative Example 4 Ingredients: 100 g osteopontin.

[0037] The effects of compositions I-VII and Comparative Examples 1-4 on calcium uptake in Caco2 cells were determined using the method described in Example 1 to characterize the potential calcium absorption-promoting effects of each composition. The results are shown in Table 2. It can be seen that the compositions containing sheep milk casein peptides, collagen tripeptides, sea cucumber peptides, and osteopontin significantly enhanced the calcium uptake capacity of Caco2 cells compared to the individual components of casein peptides, collagen tripeptides, sea cucumber peptides, and osteopontin. This indicates that casein peptides, collagen tripeptides, sea cucumber peptides, and osteopontin can synergistically promote calcium absorption. Composition VI showed the best potential calcium absorption-promoting activity; therefore, composition VI was used as the calcium absorption-promoting composition for subsequent animal experiments.

[0038] Table 2. Effects of the composition on calcium uptake in Caco2 cells

[0039] Example 3: Effect of the composition on serum calcium content in osteoporotic mice Animal grouping: Eighty-eight male C57BL / 6J mice aged 5-6 weeks were selected and housed at 23±3℃, 65±5% humidity, and a 12-hour light-dark cycle with good ventilation. The mice were housed for one week before the experiment to acclimatize to the laboratory environment.

[0040] The "triple calcium combination" administered by gavage contained calcium lactate, calcium glycinate, and calcium aspartate in a mass ratio of 1:3:2.

[0041] Mice were randomly divided into four groups: a blank control group, a model group, a triple calcium supplement group, a triple calcium supplement plus a low-dose calcium absorption-promoting composition group, a triple calcium supplement plus a medium-dose calcium absorption-promoting composition group, a triple calcium supplement plus a high-dose calcium absorption-promoting composition group, a control group 1, a control group 2, a control group 3, a control group 4, and a positive control group, with 8 mice in each group. Treatments were as follows: (1) Control group: physiological saline was administered by gavage for 30 days during the experiment, and physiological saline was injected into the peritoneum 4 hours after gavage each day.

[0042] (2) Model group: mice were given physiological saline by gavage daily for 30 days. After gavage for 4 hours each day, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW.

[0043] (3) Tricalcium combination group (calcium supplementation only, without calcium absorption promoter): For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with the tricalcium combination group and gavage was given at a dose of 19 mg / kg·BW.

[0044] (4) Tricalcium-based combination + low-dose calcium absorption-promoting composition group: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with the tricalcium-based combination + low-dose calcium absorption-promoting composition (composition VI). The calcium source was administered by gavage at a dose of 19 mg / kg·BW, and the calcium absorption-promoting composition was administered by gavage at a dose of 42 mg / kg·BW.

[0045] (5) Tricalcium-based combination + medium-dose calcium absorption-promoting composition group: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with the tricalcium-based combination + medium-dose calcium absorption-promoting composition. Calcium was administered by gavage at a dose of 19 mg / kg·BW, and the calcium absorption-promoting composition was administered by gavage at a dose of 84 mg / kg·BW.

[0046] (6) Tricalcium-based combination + high-dose calcium absorption-promoting composition group: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with the tricalcium-based combination + high-dose calcium absorption-promoting composition. Calcium was administered by gavage at a dose of 19 mg / kg·BW, and the calcium absorption-promoting composition was administered by gavage at a dose of 168 mg / kg·BW.

[0047] (7) Comparative Example 1: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with a combination of three calcium compounds + Comparative Example 1 component (sheep milk casein peptide). Calcium was administered by gavage at a dose of 19 mg / kg·BW, and sheep milk casein peptide was administered by gavage at a dose of 84 mg / kg·BW.

[0048] (8) Comparative Example 2: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with a combination of three calcium compounds + Comparative Example 2 (collagen tripeptide). Calcium was administered by gavage at a dose of 19 mg / kg·BW, and collagen tripeptide was administered by gavage at a dose of 84 mg / kg·BW.

[0049] (9) Comparative Example 3: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with a combination of three calcium compounds + Comparative Example 3 (sea cucumber peptide). Calcium was administered by gavage at a dose of 19 mg / kg·BW, and sea cucumber peptide was administered by gavage at a dose of 84 mg / kg·BW.

[0050] (10) Comparative Example 4: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, the saline was replaced with a combination of three calcium compounds + Comparative Example 4 component (osteopontin). Calcium was administered by gavage at a dose of 19 mg / kg·BW, and osteopontin was administered by gavage at a dose of 84 mg / kg·BW.

[0051] (11) Positive drug group: For the first 10 days of the experiment, mice were given saline by gavage daily. Four hours after gavage, cyclophosphamide was injected intraperitoneally at a dose of 100 mg / kg·BW. For the next 20 days, saline was replaced with calcium carbonate and administered by gavage at a dose of 19 mg / kg·BW.

[0052] Thirty days later, mouse feces were collected, and the mice were euthanized after orbital blood sampling. Femoral tissue was collected from the mice. The blood was centrifuged at 6000 g for 20 min to obtain serum, which was then diluted with physiological saline and the serum calcium content was determined using a kit.

[0053] The serum calcium levels of mice in each group were measured as follows: Figure 1 As shown in the figure, compared with the control group, the serum calcium content of mice in the model group was significantly decreased, indicating that cyclophosphamide caused calcium loss in mice, and the modeling was successful. After intervention with a triple calcium compound, triple calcium compound plus different doses of calcium absorption-promoting compounds, triple calcium compound plus different calcium absorption-promoting single components (comparative examples), and positive control drugs, the serum calcium content of mice increased significantly.

[0054] Under the same gavage dose, compared with the single grouping of comparative groups 1-4, the serum calcium content of mice in the medium-dose combination group was significantly increased, indicating that there is a synergistic effect of calcium absorption promotion among the combination of sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin.

[0055] Example 4: Effect of the composition on bone calcium content in osteoporotic mice Mouse femurs were taken, soft tissues were removed, and the samples were dried and ashed at 580°C in a muffle furnace for 8 hours. The ash was dissolved in 6 M HCl, and the digested samples were filtered and the calcium content was determined using a kit.

[0056] The results of femoral calcium levels in each group of mice are as follows: Figure 2 As shown in the figure, compared with the control group, the bone calcium content of the model group mice was significantly decreased, indicating that cyclophosphamide caused bone calcium loss in mice, leading to osteoporosis. However, after intervention with a three-calcium compound, three-calcium compound plus different doses of calcium absorption-promoting compounds, three-calcium compound plus different calcium absorption-promoting single components (comparative examples), and a positive control drug, the calcium content in the femur of mice significantly increased, indicating that different calcium absorption-promoting substances improve osteoporosis by promoting calcium entry into the blood and further transporting calcium to bone tissue. Furthermore, the bone calcium content of mice in different dose combination groups was significantly higher than that of mice in comparative examples 1-4, with medium and high dose combinations showing better effects. Under the same gavage dose, compared with the single-group groups in comparative examples 1-4, the bone calcium content of mice in the medium dose combination group was significantly increased, indicating a synergistic calcium absorption-promoting effect among the combination of sheep milk casein peptides, collagen tripeptides, sea cucumber peptides, and osteopontin.

[0057] Example 5: Effect of the composition on fecal calcium content in osteoporotic mice After drying, an appropriate amount was weighed and ashed at 580°C in a muffle furnace for 8 hours. The mixture was then digested with a mixture of HNO3 and HClO4. After filtration, the calcium content of the digest was determined using a reagent kit.

[0058] The results of fecal calcium level measurements in each group of mice are as follows: Figure 3 As shown in the figure, compared with the control group, the fecal calcium content of mice in the model group was significantly increased, indicating that cyclophosphamide caused calcium loss and excretion from the body. However, after intervention with a three-calcium compound, three-calcium compound plus different doses of calcium absorption-promoting compounds, three-calcium compound plus different calcium absorption-promoting single components (comparative example), and positive control drugs, the fecal calcium content of mice decreased significantly, indicating that the calcium absorption-promoting substances effectively prevented calcium from being excreted from the body.

[0059] The fecal calcium content of mice in different dose groups was significantly lower than that of mice in the control groups 1-4. Moreover, under the same gavage dose, the fecal calcium content of mice in the medium dose group was significantly lower than that of the control groups 1-4. This indicates that the combination of sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin synergistically inhibits the excretion of calcium in the body, thereby exerting a high calcium retention rate and improving osteoporosis.

[0060] Example 6: Effect of the composition on the expression levels of TRPV5 and TRPV6 mRNA in mouse intestinal calcium ion channel proteins The effect of the composition on the expression levels of TRPV5 and TRPV6 mRNA in mouse intestinal calcium ion channel proteins is as follows: Figure 4 As shown, compared with the control group, the expression levels of calcium channel proteins TRPV5 and TRPV6 mRNA in the jejunum of model group mice were significantly decreased, indicating that cyclophosphamide reduced the transintestinal epithelial transport of calcium ions in mice, preventing further absorption into the bloodstream. After intervention with a triple calcium-based combination, a triple calcium-based combination plus different doses of calcium absorption-promoting combinations, and a positive control drug, the expression level of TRPV5 mRNA in the jejunum of mice significantly increased, with the expression level being even higher after the addition of the calcium absorption-promoting combination. Simultaneously, the triple calcium-based combination plus different doses of calcium absorption-promoting combinations significantly increased the expression level of TPRV6 mRNA in the jejunum of mice. These results indicate that different calcium absorption-promoting substances synergistically improve the intestinal calcium transport capacity of mice, enhance the transmembrane transport capacity of calcium ions, restore calcium homeostasis, further promote bone formation, and thus improve osteoporosis.

[0061] Example 7 Effect of the composition on the expression level of Calbindin-D9k mRNA in mouse intestine The effect of the composition on the expression level of the mouse intestinal calcium-binding protein Calbindin-D9k mRNA is as follows: Figure 5As shown, compared with the control group, the expression level of calcium-binding protein Calbindin-D9k mRNA in the jejunum of model group mice was significantly decreased, indicating that cyclophosphamide weakens the binding capacity of the mouse intestine to calcium ions, leading to reduced calcium absorption and consequently lower blood calcium levels. This deficiency cannot compensate for bone calcium levels, resulting in osteoporosis. After intervention with a triple calcium-based combination, a triple calcium-based combination plus different doses of calcium absorption-promoting combinations, and a positive control drug, the level of calcium-binding protein Calbindin-D9k mRNA in the jejunum of mice significantly increased. The addition of calcium absorption-promoting combinations resulted in a higher expression level of Calbindin-D9k mRNA in the jejunum of mice, indicating that different calcium absorption-promoting substances synergistically improved the intestinal calcium absorption capacity of mice.

[0062] Example 8 Effect of the composition on mouse intestinal peptide transporter PepT1 mRNA The effect of the composition on mouse intestinal peptide transporter PepT1 mRNA is as follows: Figure 6 As shown, compared with the control group, the expression level of PepT1 mRNA in the jejunum of model group mice was significantly decreased. After intervention with a combination of three calcium-based calcium supplements and different doses of calcium absorption-promoting compounds, the level of PepT1 mRNA in the jejunum of mice was significantly increased, indicating that different calcium absorption-promoting substances can synergistically promote the transmembrane transport of peptide-bound calcium, improve the utilization rate of calcium from the intestinal lumen to the blood, and further help improve bone calcium levels, thereby improving osteoporosis.

[0063] Example 9 Effect of the composition on the mRNA expression levels of mouse intestinal sodium-calcium exchanger 1 (NCX-1) and plasma membrane calcium pump protein (PMCA1b) The effect of the composition on the mRNA expression levels of NCX-1 and PMCA1b in the mouse intestine is as follows: Figure 7 As shown, compared with the control group, the mRNA expression levels of NCX-1 and PMCA1b in the jejunum of model group mice were significantly decreased, indicating that cyclophosphamide weakens the calcium ion efflux capacity of the basolateral membrane of mouse intestinal epithelial cells, leading to intracellular calcium ion retention, ultimately reducing calcium transmembrane transport, causing hypocalcemia, and accompanied by osteoporosis. After intervention with a tricalcium-based combination, tricalcium-based combination plus different doses of calcium absorption-promoting combinations, and positive control drugs (especially the intervention with medium and high doses of the combination), the mRNA expression levels of NCX-1 and PMCA1b in the jejunum of mice were significantly increased, indicating that different calcium absorption-promoting substances can synergistically restore the basolateral calcium efflux capacity, enhance the efficiency of calcium entry into the blood, and allow more calcium to reach the bone, maintaining the body's calcium homeostasis.

[0064] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A composition that helps improve bone density, characterized in that, The composition comprises sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin, wherein the mass ratio of sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin is 20-23:9-10:35-40:30-35.

2. The composition according to claim 1, characterized in that, The mass ratio of sheep milk casein peptide, collagen tripeptide, sea cucumber peptide and osteopontin is 20:10:40:

30.

3. A product comprising the composition of claim 1 or 2, wherein the product is a health food or a pharmaceutical.

4. The product according to claim 3, characterized in that, The product also includes a calcium source.

5. The product according to claim 4, characterized in that, The calcium source includes organic calcium.

6. The product according to claim 5, characterized in that, The organic calcium is amino acid calcium.

7. The product according to claim 4, characterized in that, The calcium sources include calcium lactate, calcium glycinate, and calcium aspartate.

8. The product according to claim 7, characterized in that, The mass ratio of calcium lactate, calcium glycinate, and calcium aspartate is 1-3:1-3:1-3.

9. Use of the composition according to claim 1 or 2 in the preparation of a product that promotes calcium absorption.

10. The application according to claim 9, characterized in that, The product (1) Increase blood calcium levels; (2) Increase bone calcium levels; and / or (3) Reduce fecal calcium levels.

11. Use of the composition of claim 1 or 2 in the preparation of products that help improve bone density.

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